Throughout the application various publications are referenced in parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in the application in order to more fully describe the state of the art to which this invention pertains.
1. The Field of the Invention
This invention relates to the medical arts. In particular, it relates to a method of selectively inducing death of malignant cells in vitro and in vivo.
2. Discussion of the Related Art
There are four main types of potassium channels: inverse rectifier potassium channels (Klr); voltage-gated potassium channels (KV); calcium-activated potassium channels (Ca2+-activated K+ channel; i.e., KCa); and ATP-sensitive potassium channels (KATP) (Nelson, M. T. and Quayle, J. M., Physiological roles and properties of potassium channels in arterial smooth muscle, Am. J. Physiol. 268(4 Pt 1):C799–822 [1995]). The KCa and KATP potassium channels are ubiquitously distributed in tissues including brain capillaries. The KCa is an important regulator of cerebral blood vessel tone (Nelson M T, Quayle J M. Physiological roles and properties of potassium channels in arterial smooth muscle, Am. J. Physiol. 268(4 Pt 1): C799–822[1995]). The KCa channel is ubiquitously distributed in tissues as α and β subunits. Its activity is triggered by depolarization and enhanced by an increase in cytosolic calcium dication (Ca2+). A local increase in Ca2+ is sensed by the extremely sensitive brain α-subunit of the KCa, directed towards the cytoplasm in the cell, that allows a significant potassium cation flux through these channels.
There is growing evidence that membrane ion channels are involved in cell differentiation and proliferation. Potassium channels interfere with a variety of different cell lines derived from breast carcinoma (Wegman et al. Pfuegers Arch. 417:562–570 [1991]), melanoma (Wienhuesal et al. 151:149–157 [1996]), and neuroblastoma (Dubois B and Dubois J M. Cellular Signaling 4:333–339 [1991]). The KCa channels are known to regulate cell membrane potential and, thus, may have a role in cell proliferation. Biochemical modulation of KCa channels induces K+ flux causing membrane hyperpolarization affecting the entry of calcium dication. Excessive K+ conductance causes reduction in membrane potential, induces cell death by apoptosis or necrosis in hypoxia and ischemia. Brain tumor cells appear to express immunopositive KCa channels as studied immunohistochemically with polyclonal anti-KCa antibody. Further, others have shown that KCa channels expressed on human glioma cells are highly sensitive to [Ca2+]i concentration. However, the effect of KCa channel activation in glioma cell proliferation has not been so far studied.
Treatments directed to the use of potassium channel activators or agonists have been taught for disorders including hypertension, cardiac and cerebral ischemia, nicotine addiction, bronchial constriction, and neurodegenerative diseases, and for increasing the permeability of the blood brain barrier. (Erhardt et al., Potassium channel activators/openers, U.S. Pat. No. 5,416,097; Schohe-Loop et al., 4, 4′-bridged bis-2, 4-diaminoquinazolines, U.S. Pat. No. 5,760,230; Sit et al., 4-aryl-3-hydroxyquinolin-2-one derivatives as ion channel modulators, U.S. Pat. No. 5,922,735; Garcia et al., Biologically active compounds, U.S. Pat. No. 5,399,587; Cherksey, Potassium channel activating compounds and methods of use thereof, U.S. Pat. No. 5,234,947).
Apoptosis is programmed cell death, as signaled by the nuclei in normally functioning human and animal cells, when age or state of cell health and condition dictates. Apoptosis is an active process requiring metabolic activity by the dying cell, often characterized by cleavage of the DNA into fragments that give a so called laddering pattern on gels. Cancerous cells, however, are typically unable to experience the normal cell transduction or apoptosis-driven natural cell death process. Consequently, mechanisms have been sought by which apoptosis may be induced in malignant cells.
Mechanisms of induction of apoptosis in several different cell types and under various physiological conditions have been at least partially studied, and some apoptotic mechanisms appear to be mediated by complex signal transduction pathways involving the phosphorylation and/or dephosphorylation of signal transducing peptides. For example, phosphotyrosine phosphatase inhibitors or activators of protein tyrosine kinase induced apoptosis in B- and T-lymphocytes. (Schieven, G. L., Phosphotyrosine phosphatase inhibitors or phosphotyrosine kinase activators for controlling cellular proliferation, U.S. Pat. No. 5,877,210; Schieven, G. L., Use of phosphotyrosine phosphatase inhibitors for controlling cellular proliferation, U.S. Pat. No. 5,693,627). Also, expression of cytoplasmic Bruton's tyrosine kinase (BTK) has been linked to apoptosis in some cell lines. (Islam, T. C. et al., BTK mediated apoptosis, a possible mechanism for failure to generate high titer retroviral producer clones, J. Gene Med. 2(3):204–9 [2000]). On the other hand, signaling by activated Signal Transducers and Activators of Transcription (STATs) may participate in oncogenesis by stimulating cell proliferation and preventing apoptosis. (E.g., Bowman, T. et al., STATs in oncogenesis, Oncogene 19(21):2474–88[2000]; Reddy, E. P., et al., IL-3 signaling and the role of Src kinases, JAKs and STATs: covert liason unveiled, Oncogene 19(21):2532–47 [2000]).
Some hypothetical apoptotic mechanisms may be mediated by the activity of certain varieties of potassium channel, but contrary and varied effects indicate that different potassium channels might play different and specific mechanistic roles in apoptosis, if they play any direct role at all. For example, the Kv1.3 voltage-gated potassium channel has been implicated in the pathway for Fas-induced apoptosis. (E.g., Gulbins, E. et al., Ceramide-induced inhibition of T-lymphocyte voltage-gated potassium channel is mediated by tyrosine kinases, Proc. Natl. Acad. Sci. USA 94(14):7661–6 [1997]). Expression of Kir1.1 potassium channel from an expression vector caused apoptosis in hippocampal neurons. (Nadeau, H. et al., ROMK1 (Kir1.1) cause apoptosis and chronic silencing of hippocampal neurons, J. Neurophysiol. 84(2):1062–75 [2000]). Also, tumor necrosis factor (TNF)-α-mediated apoptosis of liver cells was dependent on activation of unspecified potassium channels and chloride channels and was further dependent on the presence of calcium dication and protein kinase C activity. (Nietsch, H. H. et al., Activation of potassium and chloride channels by tumor necrosis factor alpha, J. Biol. Chem. 275(27):20556–61 [2000]).
In contrast, the KATPpotassium channel activator cromakalim prevented glutamate-induced or glucose/hypoxia-induced apoptosis in hippocampal neurons. (Lauritzen, I. et al.,The potassium channel opener (−)-cromakalim prevents glutamate-induced cell death in hippocampal neurons, J Neurochem, 69(4):1570–9 [1997]). Clofilium, an inhibitor of the Kv1.5 delayed rectifier potassium channel, induced apoptosis of human promelocytic leukemia (HL-60) cells. (Choi, B. Y. et al., Clofilium, a potassium channel blocker, induces apoptosis of human promelocytic leukemia (HL-60) cells via Bcl-2-insensitive activation of caspase-3, Cancer Lett, 147 (1–2):85–93 [1999]; Malayev, A. A. et al., Mechanism of clofilium block of the human Kv1.5 delayed rectifier potassium channel, Mol. Pharmacol. 47(1):198–205 [1995]). Also, Kv inhibitor 4-aminopyridine induced apoptosis in HepG2 human hepatoblastoma cells. (Kim, J. A. et al., Ca2+ influx mediates apoptosis induced by 4-aminopyridine, a K+ channel blocker, HepG2 human hepatoblastoma cells, Pharmacology 60(2):74–81 [2000]).
Thus, links between various types of potassium channels and any particular mechanisms of apoptosis remain unclear, and no role for calcium-activated potassium channels (KCa), in particular, has been suggested.
The ability to induce apoptosis in malignant cells would be especially desirable with respect to malignant tumors, especially tumors of the central nervous system. These malignancies are usually fatal, despite recent advances in the areas of neurosurgical techniques, chemotherapy and radiotherapy.
The glial tumors, or gliomas, comprise the majority of primary malignant brain tumors. Gliomas are commonly classified into four clinical grades, with the most aggressive or malignant form of glioma being glioblastoma multiforme (GBM; also known as astrocytoma grade IV), which usually kills the patient within 6–12 months. (Holland, E. C. et al., Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice, Nat. Genet. 25(1):55–57 [2000]; Tysnes, B. B et al., Laminin expression by glial fibrillary acidic protein positive cells in human gliomas, Int. J. Dev. Neurosci. 17(5-6):531–39 [1999]).
GBM tumors are characterized by rapid cell growth and extensive invasion into the surrounding normal brain tissue. GBM tumors are difficult to remove surgically and typically recur locally at the site of resection, although metastases also may occur within the central nervous system. Tumor cell movement within the central nervous system is a complex process that involves tumor cell attachment to the extracellular matrix (ECM) via cell surface receptors, degradation of the ECM by proteolytic enzymes, including serine proteases and matrix metalloproteinases, and subsequent tumor cell locomotion. (Tysnes et al. [1999]; MacDonald, T. J. et al.,Urokinase induces receptor mediated brain tumor cell migration and invasion, J. Neurooncol. 40(3):215–26 [1998]; Mäenpää, A. et al., Lymphocyte adhesion molecule ligands and extracellular matrix proteins in gliomas and normal brain: expression of VCAM-1 in gliomas, Acta Neuropathol. (Berl.) 94(3):216–25 [1997]). Thus, malignant gliomas overexpress members of the plasminogen activator system and characteristically invade by migrating on ECM-producing white matter tracts and blood vessel walls. (Tysnes et al. [1999]; Colognato, H. and Yurchenco, P. D., Form and function: the laminin family of heterotrimers, Dev. Dyn. 218(2):213–34 [2000]).
Despite a wealth of molecular biological, biochemical and morphological information that is available today on gliomas, the prognosis with treatment has not significantly changed in the last two decades and remains among the worst for any kind of malignancy. (E.g., Shapiro, W. R., Shapiro, J. R., Biology and treatment of malignant glioma, Oncology 12:233–40 [1998]; Thapar, K. et al., Neurogenetics and the molecular biology of human brain tumors, In: Brain Tumors, Edit. Kaye A H, Laws E R, pp.990. [1997]). In particular, there are no standard therapeutic modalities that can substantially alter the prognosis for patients with malignant glial tumors of the brain, cranium, and spinal cord. Although intracranial tumor masses can be debulked surgically, treated with palliative radiation therapy and chemotherapy, the survival associated with intracranial glial tumors, for example, a glioblastoma, is typically measured in months.
The present invention provides a much needed method of inducing apoptosis in glioma cells, in vitro and in vivo, that employs activators of calcium-activated potassium channels. This and other benefits of the present invention are described herein.